Ubiquitin-Activated Interaction Traps (UBAITs) identify E3 ligase binding partners.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• HECT and RING E3 ubiquitin ligases play crucial roles in cellular regulation.
• Identifying their substrates, adaptors, and regulators is essential for understanding ubiquitin signaling.
• Current methods for profiling E3 ligase interactions have limitations.

Purpose of the Study:

• To develop a novel class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3 ligases.
• To demonstrate the utility of these reagents, termed Ubiquitin-Activated Interaction Traps (UBAITs), in both in vitro and cellular contexts.

Main Methods:

• Design and construction of E3-ubiquitin fusion proteins (UBAITs) for specific HECT and RING E3 ligases.
• Utilizing E1 and E2 enzymes to facilitate covalent linkage between the UBAIT's C-terminal ubiquitin and interacting proteins.
• Co-purification of E3 ligases with their binding partners.
• Expression of UBAITs in yeast and human cells to identify interacting proteins in vivo.

Main Results:

• UBAITs were successfully designed for HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3 ligases.
• In vitro experiments showed that UBAITs could trap interacting proteins via E3 or E2 thioester intermediates.
• Both wild-type and active-site mutant UBAITs successfully identified known interacting proteins in yeast and human cells.
• Expression of Psh1, RNF126, and RNF168 UBAITs in cells confirmed their ability to trap known partners.
• The RNF168 UBAIT identified histone protein H2AZ as a novel target of RNF168, implicating it in DNA repair pathways.

Conclusions:

• UBAITs represent a powerful and versatile new class of reagents for profiling E3 ubiquitin ligase interactions.
• These tools enable the covalent capture and identification of substrates, adaptors, and regulators across a wide range of E3 ligases.
• The identification of H2AZ as an RNF168 target highlights the potential of UBAITs for discovering novel E3 ligase substrates and functions in biological processes like DNA repair.